Tripterygium wilfordii Hook F extracts and components thereof for immunosuppression

ABSTRACT

The present invention involves the use of Tripterygium Wilfordii Hook F extracts in the treatment of rheumatoid arthritis. An alcohol extract of this plant (T2) inhibited antigen- and mitogen-stimulated proliferation of T cells and B cells, cell cycle progression, interleukin-2 (IL-2) production by T cells, immunoglobulin production by B cells and interleukin-2 mRNA production. T2 did not affect IL-2 receptor expression by T cells, IL-1 production by monocytes, the capacity of monocytes to present antigen, or signaling pathways. Inhibition could not be accounted for by nonspecific toxicity. These results support the conclusion that T2 exerts a powerful suppressive effect on human immune responses. Suppressing autoimmune disease is a most preferred embodiment of this invention.

The government has rights in the present invention as research relevantto the development thereof was supported by a grant from the UnitedStates government, NIH grant AR-36169.

This application is a continuation of application Ser. No. 08/136,345,filed Oct. 14, 1993, now U.S. Pat. No. 5,500,340, and acontinuation-in-part of application Ser. No. 07/494,113 filed Mar. 14,1990, now abandoned.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is a chronic inflammatory disease of uncertainetiology (1,2). Since the cause is unknown, treatment has been directedat suppressing the signs and symptoms of chronic inflammation. Althoughmany agents have been documented to decrease pain and swellingtemporarily, none has been shown to have a major impact on the course ofthe disease. New therapeutic modalities have been developed over thepast few years, employing monoclonal antibodies, cytokine antagonists,specific receptor targeted toxins, and other biologics (3-17).Nevertheless, uniform and persistent suppression of disease activity hasnot been reported. Although these approaches remain promising,alternative means of drug development seem warranted and could yield notonly new and effective treatment modalities, but also provide newinsights into disease pathogenesis that could serve as the basis offuture therapeutic innovations.

An area to search for new therapeutic interventions for RA is that oftraditional Chinese medicines. One of these traditional medicines isfrom Tripteryguim wilfordii Hook F, a shrub-like vine from theCelastraceae family (18). A variety of preparations derived from thisplant have been used in South China for many years to treat differentforms of arthritis and other autoimmune diseases. In 1978, an extract ofTripterygium wilfordii Hook F was produced by chloroform methanolextraction of the woody portion of the roots and designated T2 (18).Reports in the Chinese literature describe T2 treatment of more than 750patients with a variety of autoimmune diseases (19-35). The generalimpression has been that T2 is well-absorbed orally, appears to haveacceptable toxicity, and is effective in the treatment of variousautoimmune diseases.

T2 was evaluated in a double-blind placebo controlled cross-over studyinvolving 70 RA patients, with a mean disease duration of 6 years(19-20). Statistically significant improvement in all clinicalparameters, particularly ESR, CRP, and Rheumatoid factor titers, wasnoted after 12 weeks of therapy in the experimental group compared witheither baseline measurements or the placebo treated group. Of thepatients treated, 82-93% noted improvement in different clinicalcriteria or laboratory correlates of inflammation. An immunosuppressiveactivity was implicated by the finding that treatment induced inhibitionof the production of IgM and IgM rheumatoid factor by the patients'peripheral blood mononuclear cells in vitro (20). Toxicity, whichconsisted primarily of skin rash, gastrointestinal complaints andamenorrhea, was generally mild and reversible with cessation of therapy.These results support the contention that T2 might be an effectivetherapy for RA, but there is little experience with this agent outsideof China.

Tripteryguim wilfordii Hook F is known to contain a number ofconstituents, some of which appear to be toxic (36). It is known thatthe leaves, stem, flowers, and the skin of the roots are poisonous andthat ingestion can cause death (37-39). In contrast, the woody portionof the roots of the plant is much less toxic. T2 is prepared from thewoody portion of the roots, appears to contain the therapeuticcomponents, and to have reduced toxicity compared with otherpreparations.

The Chinese experience has suggested that a daily dosage of 0.8-1.5mg/kg of T2 is safe and effective. Acute and chronic toxicity studieshave been carried out in China using a variety of animal models. TheLD₅₀ in mice is 159.7±14.3 mg/kg (40). The major chronic toxicity notedin rats administered 30 mg/kg for 90 days was azoospermia and decreasein testicular weight (40). Lower dosages of T2 did not cause decreasesin testicular weight. The toxicity studies, therefore, suggest that T2exhibits a reasonable safety index and should be able to be administeredto patients safely.

Research has begun in China to determine the spectrum of activity of T2.Triptonide and triptolide from this plant have been reported to inhibitthe proliferation of lymph cells induced by concanavalin A. ((Zhang etal., Shanghai Yike Da ue Xuebao, 1986, 13 (4) pp. 267-272.))Additionally, ancient Chinese medical books have suggested that thisherbal remedy is useful to treat joint pain. Recently, this extract hasbeen used in the treatment of rheumatic diseases including rheumatoidarthritis, as well as systemic lupus erythematosus, Behcet's disease,and psoriasis. Alcoholic extracts (T2) of the plant have been describedas having significant activity in vivo against certain mouse leukemiasand in vitro against cells derived from human carcinomas (Kupchan etal., J. Am. Chem. Soc., 1972, 94 pp. 3194-3195). The capacity of T2 tosuppress a number of animal models of autoimmune disease, includingadjuvant arthritis and experimental allergic encephalomyelitis, has beenreported (41-47). Large concentrations of T2 (30 mg/kg) suppress delayedtype hypersensitivity reactivity in mice and may also suppress graftversus host disease, as well as skin and heart allograft rejection(36,41). In general, however, only very large concentrations of T2 havebeen examined in these studies. It, therefore, remains unclear whetherlower, more pharmacologically appropriate concentrations would alsoexert therapeutic effects in these animal models.

T2 is a crude extract containing a mixture of materials, includingvarious glycosides, alkaloids, and diterpenoids. The active principle,however, has not yet been identified. A few components have beenpurified, including triptolide, wilfordine, and related compounds, butproof that a particular purified component accounts for the therapeuticor immunosuppressive activity of T2 does not exist (48).

High concentrations of triptolide were reported to suppress B and Tlymphocyte proliferation and interleukin-2 production by mouse spleencells (Pu et al. (1990) Chem. Abstracts, 112:45, abstract 171972d). Theconcentrations used were sufficiently high that significant nonspecifictoxicity undoubtedly occurred.

SUMMARY OF THE INVENTION

The present invention involves the use of Tripterygium wilfordii Hook Fextracts (T2) or components thereof to selectively suppress the immunityof an animal or a patient in need of such treatment. Immunity of ananimal may include immunoglobulin synthesis, cell proliferation ofperipheral blood lymphocytes, cellular immune responses or proliferationof T and B lymphocytes.

In particular, the selective inhibition of interleukin-2 production byinhibition of IL-2 gene transcription and consequent inhibition of IL-2specific mRNA production without substantial cellular toxicity is anaspect of the present invention. Lack of substantial cellular toxicityis indicated by substantially unchanged interleukin-2 receptorexpression or cellular signaling activities such as inositoltriphosphate production, diacylglycerol generation, translocation ofprotein kinase C or protein tyrosine kinase activity. Lack ofsubstantial cellular toxicity by T2 may also mean having little or noeffect on the capacity of monocytes to function as antigen presentingcells, having little or no effect on the growth of endothelial cells orfibroblasts, or having little or no effect on the viability of eitherresting or stimulated lymphocytes, endothelial cells, fibroblasts,monocytes or polymorphonuclear leukocytes.

The administration of Tripteryguim wilfordii Hook F T2 extract in atherapeutically effective amount to suppress autoimmune disease in apatient in need of such treatment is a most preferred embodiment of thisinvention. A therapeutically effective amount of T2 inhibits IL-2production without substantial cellular toxicity. An in vivotherapeutically effective amount of T2 for humans is about 60 mg/day. Anin vitro therapeutically effective amount of T2 for cell cultures isabout 1.0 μg/ml. Particular autoimmune diseases thought amenable to suchtreatment include rheumatoid arthritis, systemic lupus erythematosus andpsoriasis.

A method of testing for selective inhibition of IL-2 specific mRNAproduction is an aspect of the present invention, the method consistingessentially of: culturing eukaryotic cells in culture with andseparately without Tripterygium wilfordii Hook F T2 extract orcomponents thereof in a therapeutically effective amount to provide atest sample and a control sample; measuring IL-2 mRNA level and areference mRNA level such as actin mRNA to provide a test IL-2 mRNAsample, a test reference mRNA sample, a control IL-2 mRNA sample and acontrol reference mRNA sample; comparing (test IL-2 mRNA level+controlIL-2 mRNA level) to (test reference mRNA level+control reference mRNAlevel); wherein when (test IL-2 mRNA level+control IL-2 mRNA level) issubstantially less than 1 and (test reference mRNA level+controlreference mRNA level) is about 1, selective inhibition of IL-2 mRNAproduction by T2 is indicated.

The specific T2 impairment of IL-2 mRNA production with nontoxicity toother cellular functions and other cell types is a surprising andunexpected aspect of the present invention. This specificity of T2 canbe ascribed to individual or a combination of components of the T2extract.

ABBREVIATIONS

CRP=cross-reacting protein

DAG=diacylglycerol

ESR=erythrocyte sedimentation rate

FACS=fluorescence-activated cell sorter

Ig=immunoglobulin

IL-2=interleukin-2

IL-2R=interleukin-2 receptor

IP=phosphatidyl inositol triphosphate

MAb=monoclonal antibodies

NHS=normal human serum

PBMC=peripheral blood mononuclear cells

PDB=phorbol dibutyrate

PHA=phytohemagglutinin

PKC=protein kinase C

RA=rheumatoid arthritis

SA=formalinized Staphylococcus aureus

SK=streptokinase

SRBC=sheep red blood cells

T2=an ethanol extract from the woody portion of Tripteryguim wilfordiiHook F

TT=tetanus toxoid

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of T2 on T cell proliferation. T cells (1×10⁵ /well) werecultured with medium (□) or PHA (Δ) in the presence or absence ofvarying concentrations of T2 as indicated for 3 days. Results representthe mean cpm±SEM of three experiments.

FIG. 2. Effect of T2 on cell cycle progression of human T cells. T cells(1×10⁵ /well) were cultured with or without PHA (1 ug/ml) in the absenceor presence of the indicated concentrations of T2 for 24, 48 or 72 hrs.The samples were harvested, stained with acridine orange, and analyzedwith an ORTHO flow cytometer using the CICERO program to determine theposition of cells in the cell cycle as assessed by their RNA and DNAcontent.

FIG. 3. Inhibitory effect of T2 on IL-2 production. T cells (1×10⁵/well) were cultured with medium (□) or PHA (▪) in the presence orabsence of varying concentrations of T2 for 36 hrs. The cell-freesupernatants were diluted 1:4 and analyzed for IL-2 activity with CTLL-2cells. Mean ³ H!-TdR incorporation±SEM of CTLL-2 cells from 6experiments is shown.

FIG. 4. Effect of supplemental IL-2 on T2 mediated inhibition of T cellproliferation. T cells (1×10⁵ /well) were stimulated with PHA with (□)or without (▪) IL-2 (10 U/ml) and in the presence or absence of varyingconcentrations of T2 for 3 days. The data are expressed as percent ofcontrol ³ H!-TdR incorporation from three experiments.

FIG. 5. Effect of T2 on steady state levels of IL-2 mRNA in mitogenstimulated T cells. T cells (1×10⁶ /ml) were cultured with and withoutPHA in the presence or absence of T2 (1 μg/ml). After a 4-hourincubation, total RNA was isolated and IL-2 and actin mRNA levelsdetermined by S1 nuclease protection as described (54).

FIG. 6. Effect of T2 on B cell DNA synthesis and Ig production. In theleft panel, B cells (5×10⁴ /well) were stimulated with SA (∘) or SA+IL-2(∘), and in the right panel with SA+IL-2 in the presence of varyingconcentrations of T2. ³ H!-TdR was determined after a 5-day incubation(left panel). Supernatants were harvested after a seven-day culture andassayed for IgM (▪), IgG (∘) and IgA (∘) content (right panel). Resultsare the mean±SEM of 3 experiments.

FIG. 7. Effect of T2 on total IP generation by activated T cells. FreshT cells (A) or Jurkat cells (B) were labeled with ³ H!-myo-inositolovernight in the absence or presence of the indicated concentrations ofT2. Total IP was determined as described in Example 2. An aliquot ofeach cell population was also stimulated with PHA for 24 hours andsupernatants assayed for IL-2 content using CTLL-2 cells (∘). Data arethe mean of three replicate experiments.

FIG. 8. Effect of T2 on the generation of IP fractions by PHA activatedT cells. Jurkat cells were labeled with ³ H!-myo-inositol overnight inthe presence or absence of various concentrations of T2. Following a 5minute incubation with 10 mM LiCl, the cells were activated with PHA for60 min. Water soluble IPs were isolated (IP1, top panel; IP2, middlepanel and IP3, bottom panel) and quantitated as described in Example 2.Data are from one of three similar experiments.

FIG. 9. Effect of T2 on DAG generation and IL-2 secretion by PHAstimulated T cells. DAG and IL-2 were assayed as described in Example 2.Data represent the mean of duplicate determination of three similarexperiments.

FIG. 10. Effect of T2 on translocation of PKC. PKC activity in both thecytoplasmic and membrane fractions were assayed as described in Example2.

FIG. 11. Effect of T2 on protein tyrosine phosphorylation. See Example 2for methods. Arrows indicate tyrosine phosphorylation of new proteinsafter PHA stimulation.

FIG. 12 summarizes assessment of symptomatic improvement in rheumatoidarthritis patients as a result of treatment with a mixture fromTripteryguim wilfordii Hook F.

FIG. 13 schematically shows the structure of triptolide (1) andtriptodiolide (2).

FIG. 14 schematically shows the structure of triptonide.

FIG. 15 schematically describes the structure of wilfortrine (1) andwilfortrine methyl ester (2).

FIG. 16 shows the structure of triptophenolide (1) and triptophenolidemethyl ester (2).

FIG. 17 schematically shows the structure of triptonoterpenol.

FIG. 18 schematically shows the structure of wilformine.

FIG. 19 outline the extraction procedure for preparation of triptolide.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention concerns the use of Tripterygium wilfordii Hook Fextracts to suppress immune function, particularly for the treatment ofautoimmune diseases. T2 was prepared by ethanol extraction ofTripteryguim wilfordii Hook F roots.

Example 1 concerns studies on the effect of T2 on human lymphocytefunction. Interleukin-2 production by T cells is inhibited by T2 due toan inhibition of gene transcription while the expression of IL-2receptors is not affected. T2 also suppresses proliferation of B cellsand immunoglobulin production by B cells. The experiments described inExample 2 indicate that signaling pathways are not affected by T2demonstrating the selective nature of T2 inhibition. Example 3 concernsresults of studies on the effect of T2 in the treatment of patients withrheumatoid arthritis.

EXAMPLE 1 Effect of T2 on Human Lymphocyte Function

This example describes the effect of T2 on in vitro immuneresponsiveness of human peripheral blood mononuclear cells (PBMC)obtained from normal individuals. It was found that T2 exerted aconcentration-dependent profile of suppressive activity on both T celland B cell functions, whereas the functional activities of monocyteswere more resistant to the suppressive effects of T2.

Methods

Cell preparation. PBMC were obtained from the blood of healthy adults bycentrifugation on sodium diatrizoate/Ficoll gradients (Sigma, St. Louis,Mo.). Monocytes were isolated from PBMC by centrifugation onSepra-cell-MN (Sepratech, Oklahoma City, Okla.) or by glass adherence.The monocytes obtained from the two procedures were used to examineinterleukin-1 (IL-1) production and antigen presentation, respectively.For purification of T cells and B cells, PBMC were incubated withL-leucine methyl ester HCl (Sigma) for 45 minutes at room temperature todeplete monocytes and natural killer cells, (Thiele et al., J. Immunol.131:2282-2290, 1983). The resultant lymphocytes were rosetted withneuraminidase-treated sheep red blood cells (SRBC) and were thenseparated by Ficoll/diatrizoate centrifugation, (Rosenberg, et al., J.Immunol. 122:926-931, 1979). T cells were further purified by passage ofthe rosette-positive population over a nylon-wool column to removeresidual B cells and monocytes, (Rosenstreich, et al., J. Exp. Med.134:1170-1186, 1971). B cells were prepared from the initial populationof rosette-negative cells by removing any remaining cells that formedrosettes with neuraminidase-treated SRBC.

Staining with monoclonal antibodies (MAb) to CD3 and CD20 and analysiswith the fluorescence-activated cell sorter (FACS) indicated that the Tcell and B cell populations were more than 96% and 90% pure,respectively. T cells were incubated with mitomycin c (0.1 mg/ml) for 45minutes and then washed thoroughly, (Jelinek et al., J. Immunol.136:83-92, 1986).

Reagents, T2, an ethanol extract from the woody portion of the roots ofTWH, was a kind gift of Taizhou Pharmaceutical Company (Taizhou, JiangSu, People's Republic of China). It contained more than 8 differentcompounds including glycosides, diterpenoids, alkaloids, and ketones.Before use, the extract was dissolved in DMSO and further diluted withculture medium. Phytohemagglutinin (PHA; Wellcome Reagents, ResearchTriangle Park, N.C.), phorbol dibutyrate (PDB; Sigma), ionomycin(Calbiochem, San Diego, Calif.), and the anti-CD3 MAb, 64.1, were usedfor T cell activation (Geppert et al., J Immunol 138:1660-1666, 1987).MAb 64.1 was purified as previously described Hansen et al., "T cellprotocol", Leukocyte Typing. Edited by Bernard, et al. Berlin,Springer-Verlag, 1982!. Human recombinant interleukin-2 (rIL-2; Cetus,Emeryville, Calif.) and/or formalinized Staphylococcus aureus (SA;Calbiochem) was used for B cell activation. The MAb against the α chainof the IL-2 receptor (IL-2R), anti-Tac, was the gift of Dr. ThomasWaldmann (NIH, Bethesda, Md.) and was used to analyze IL-2R expression.Interleukin-1 (Cistron Technology, Pine Brook, N.J.) was purchased forstandardization of the IL-1 assay. Affinity-purified goat anti-humanIgA, IgG, and IgM and similar antibodies conjugated to horseradishperoxidase were purchased from Tago (Burlingame, Calif.). Streptokinase(SK) and tetanus toxoid (TT) were purchased from Hoechst-Roussel(Somerville, N.J.) and MCDC Biologics (Jamaica Plain, Mass.),respectively.

Cell culture and assay of lymphocyte DNA synthesis. T cells (1×10⁵/well) or B cells (5×10⁴ /well) alone or B cells with mitomycinc-treated T cells (1×10⁵ /well) were cultured in RPMI 1640 medium(Hazleton Biologics, Lenexa, Kans.) supplemented with 10% fetal calfserum, penicillin G (200 units/ml), gentamicin (10 μg/ml), andL-glutamine (0.3 mg/ml) in 96-well microtiter plates in a total volumeof 200 μl, with or without the stimuli indicated, and in the presence orabsence of various concentrations of T2. The final concentration of DMSOin culture was 0.02-0.002%. This concentration of DMSO had no effect onany of the responses analyzed.

For both T and B cell activation, immobilized anti-CD3 (MAb 64.1)stimulation was used. This MAb was immobilized by incubating 50 μl (5μg/ml) in each well for at least 2 hours at room temperature. The excesssoluble antibody was removed before cell culture (Hansen, supra). Cellswere cultured for the indicated duration, and then pulsed with 1 μCi of³ H-thymidine, (³ H-TdR; New England Nuclear, Boston, Mass.) for thelast 12 and 18 hours for T cell and B cell cultures, respectively. ³H-TdR uptake was measured in a liquid scintillation counter. All dataare expressed as the mean counts per minute of 3 replicatedeterminations (Davis et al., J Immunol 137:3758-3767, 1986).

IL-1 production assay. Monocytes (1×10⁵ /well) were suspended in RPMI1640 medium with 1% normal human serum (NHS) and cultured with orwithout lipopolysaccharide (10 μg/ml) in the presence or absence ofvarious concentrations of T2 for 24 hours. The culture supernatants werecollected, and serial dilutions were assayed for IL-1 using C3H/HeJmurine thymocytes as described elsewhere (Moreno et al., J Immunol136:3579-3587, 1986). Concentrations of T2 contained in the dilutions ofsupernatants had no effect on DNA synthesis by C3H/HeJ thymocytes.

IL-2 production assay. T cells (1×10⁵ /well) were incubated with orwithout PHA (1 μg/ml) or immobilized anti-CD3 in the presence or absenceof various concentrations of T2 for 24 hours. Cell-free supernatantswere harvested, serial dilutions were made, and IL-2 content was assayedwith CTLL-2 cells as described previously (Gillis et al., J Immunol120:2027-2032, 1978).

IL-2R expression. T cells were cultured with or without the indicatedstimuli in the presence or absence of various concentrations of T2 for36 hours. After washing, the cells were stained with saturatingconcentrations of anti-Tac or a mouse IgG control MAb, followed byfluorescein isothiocyanate-conjugated goat anti-mouse Ig antibody(Cappel, West Chester, Pa.). The samples were fixed with 1%paraformaldehyde and analyzed with a FACSTAR (Becton Dickinson, MountainView, Calif.) flow cytometer, using a single-histogram statisticsprogram (Davis, supra).

Measurement of Ig synthesis. The amount of IgG, IgA, and IgM in theculture supernatants of B cells stimulated with SA plus rIL-2 in thepresence or absence of T2 for 7 days was determined using anisotype-specific enzyme-linked immunosorbent assay method. Quantitationof the Ig in the supernatants was then determined by comparison with astandard curve. The sensitivity of the assay is 15 ng/ml for IgA andIgG, and 30 ng/ml for IgM (Splawski et al., J Immunol 139:1432-1437,1986).

Results

Effect of T2 on human T cell responsiveness. These experiments notedthat T2 caused concentration dependent inhibition of PHA induced ³H-thymidine incorporation by purified human T lymphocytes (FIG. 1).Fifty percent inhibition was noted at concentrations of approximately0.2 μg per ml. Cell cycle analysis indicated that T2 prevented cellsfrom progressing through the G1 phase of the cell cycle (FIG. 2).Mitogen induced IL-2 production by purified T Cells was also inhibitedby a similar concentration of T2 (FIG. 3). Mitogen induced expression ofIL-2 receptors was not inhibited by T2 (Table I) indicating that T2 isnontoxic to this cellular activity. These results suggested that thedecrease in proliferation might be the result of inhibition of IL-2production.

                  TABLE 1    ______________________________________    EFFECT OF T2 ON INTERLEUKIN-2 (IL-2) RECEPTOR    EXPRESSION*            Nil                  PHA                     Fluorescence       Fluorescence    T2      % positive                     intensity   % positive                                        intensity    ______________________________________    0 μg/ml            10 ± 2                     483 ± 18 65 ± 17                                        561 ± 166    0.65 μg/ml            --       --          60 ± 22                                        519 ± 109    1.25 μg/ml             9 ± 2                     504 ± 29 61 ± 20                                        525 ± 128    2.50 μg/ml            --       --          --     --    ______________________________________     *T cells (1 × 10.sup.5 /well) were cultured with medium or     phytohemagglutinin (PHA) in the presence or absence of various     concentrations of T2 as indicated for 36 hours. Cells were collected,     stained with antiTac monoclonal antibody followed by fluorescein     isothiocyanateconjugated goat antimouse IgG, and analyzed by flow     cytometry. Values are the mean ± SEM of 6 experiments.

In order to examine IL-2 production, experiments were carried out inwhich the effect of T2 on proliferation was examined in the presence ofsupplemental IL-2. As can be seen in FIG. 4, much of the inhibitoryeffect of T2 was overcome by supplemental IL-2. These results suggestedthat one of the major actions of T2 was to inhibit IL-2 production. Thisappeared to result from an inhibition of IL-2 gene transcription sinceT2 inhibited the appearance of mRNA for IL-2, as shown in FIG. 5. Theseexperiments confirmed that one action of T2 was to inhibit IL-2production.

Effect of T2 on Human B Lymphocyte Responses

Additional effects of T2 were demonstrated when its action on human Bcell responses was examined. As can be seen in FIG. 6, T2 inhibited bothmitogen-induced proliferation of highly purified B cells, as well asimmunoglobulin production in a concentration dependent manner. Theseresults suggested that T2 had additional effects beyond altering IL-2production. Some specificity for the action of T2 was demonstrated,however, when its effects on a number of other cell types were examined.Thus, T2 had no effect on IL-1 production by human monocytes nor ontheir capacity to function as antigen presenting cells (49). Inaddition, T2 had no effect on the growth of endothelial cells orfibroblasts during a 48 hour culture. None of the inhibitory effects ofT2 could be accounted for by non-specific toxicity, as inhibitoryconcentrations of T2 had no effect on the viability of either resting orstimulated lymphocytes, endothelial cells, fibroblasts, monocytes, orpolymorphonuclear leukocytes. These results support the contention thatT2 has a limited spectrum of immunosuppressive activity which cannot beaccounted for by non-specific toxic effects. Of importance, the capacityof T2 to suppress both IL-2 production by T cells, and proliferation andimmunoglobulin production by B cells could well explain the action ofthis agent in patients with RA.

EXAMPLE 2 Effect of T2 on Critical Signaling Pathways

The mechanism by which T2 might inhibit IL-2 production was examined ingreater detail. The possibility was explored that T2 might inhibit acritical signaling pathway involved in inducing transcription of theIL-2 gene. Current information suggests that T cell receptor occupancyleads to activation of tyrosine kinases, followed by stimulation ofphospholipase C. This results in production of phosphatidyl inositoltriphosphate and diacylglycerol, that induce increases in intracellularcalcium and activation of protein kinase C, respectively (50, 51, 52).Therefore, experiments were carried out to examine the possibility thatT2 might inhibit one of these signaling pathways.

Methods

Effect of T2 on total IP generation by activated T cells. Fresh T cells(A) or Jurkat cells (B) were labeled with ³ H!-myo-inositol overnight inthe absence or presence of the indicated concentrations of T2. The cellswere washed and incubated with 10 mM LiCl for 5 minutes then activatedwith PHA for 60 min. The cells were extracted with 0.75 ml of a 1:1mixture of chloroform and methanol, followed by 0.25 ml of chloroformand 0.25 ml of water. The phases were separated by centrifugation andthe water soluble fractions were applied to a 0.25 ml Agl-X8 formate ionexchange column. Total inositol phosphate was eluted with 1.5 ml of 0.1M formic acid and 1 M sodium formate. The radioactivity was quantifiedby scintillation counting. An aliquot of each cell population was alsostimulated with PHA for 24 hours and supernatants assayed for IL-2content using CTLL-2 cells.

Effect of T2 on the generation of IP fractions by PHA activated T cells.Jurkat cells were labeled with ³ H!-myo-inositol overnight in thepresence or absence of various concentrations of T2. Following a 5minute incubation with 10 mM LiCl, the cells were activated with PHA for60 min. Water soluble IPs were isolated and quantitated. To accomplishthis, the cultures were extracted with 0.75 ml of a 1:1 mixture ofchloroform/methanol, followed by 0.25 ml each of chloroform and water.The phases were separated by centrifugation and the water solublefraction was applied to a 0.25 ml Agl-X8 formate ion-exchange column,and washed extensively with 5 mM cold myo-inositol. IP1, IP2 and IP3were sequentially eluted with 4 ml of 0.2 M ammonium formate plus 0.1 Mformic acid, 10 ml of 0.4 M ammonium formate plus 0.1 M formic acid and10 ml of 1 M ammonium formate plus 0.1 M formic acid respectively. Theradioactivity of the various elution fractions was quantified byscintillation counting.

Effect of T2 on DAG generation and IL-2 secretion by PHA stimulated Tcells. T cells for each sample were cultured overnight with PHA in thepresence or absence of the indicated concentrations of T2. The cellpellets were lysed with a mixture of chloroform and methanol, andfractions separated with 1 M NaCl and chloroform. The organic phase wascollected and dried under nitrogen. DAG mass in the organic extract wasassayed by solubilizing the lipid residues in a mixture of ³² P-γ-ATPand DAG kinase and phosphatidic acid, incubating at 37° C. for 1 hourduring which DAG was quantitatively converted to p-phosphatidic acid.The samples were dried and redissolved in chloroform. The solvent wasapplied to a silica gel and separated by thin layer chromatography withchloroform/methanol/acetic acid. After visualization with iodine, thespot which contained phosphatidic acid was harvested and radioactivitydetermined by liquid scintillation counting. An aliquot of cells wasalso stimulated with mitogen and supernatants harvested after 24 hoursand assayed for IL-2 content.

Effect of T2 on translocation of PKC. Jurkat cells (1×10⁶ /ml) wereincubated overnight with or without T2 at the indicated concentrations.The cells were lysed by sonication and then cytoplasmic and membranefractions separated by centrifugation. PKC activity in both thecytoplasmic and membrane fraction was assayed using a protein kinase Cassay system (Amersham) which employed a synthetic peptide as aphosphate acceptor in the presence of phosphatidylserine, calcium andPMA.

Effect of T2 on protein tyrosine phosphorylation. Jurkat cells (3×10⁶)were incubated overnight in the absence or presence of the indicatedconcentrations of T2. The cells were washed and stimulated with PHA for30 minutes. After centrifugation, the pelleted cells were solubilizedwith 1×SDS sample buffer containing protease inhibitors. The lysateswere centrifuged at 10,000 rpm for 15 minutes. The supernatants wereanalyzed for protein phosphorylation by western blotting using a mousemonoclonal antibody (Upstate Biotechnology, Inc.) againstphosphotyrosine.

Results

The effect of T2 on mitogen induced production of phosphatidyl inositolmetabolites. As can be seen in FIG. 7, mitogenic stimulation lead to theproduction of IL-2 and phosphatidyl inositol metabolites. Whereas IL-2production was inhibited, generation of phosphatidyl inositolmetabolites was not. Similar results were seen in fresh T cells and inthe Jurkat leukemic T cell line. Additional experiments examined whetherT2 specifically inhibited generation of IP3, which is thought to induceincreases in intracellular calcium (52). As can be seen in FIG. 8, T2had no effect on the generation of IP3 or other specific PI metabolitesby mitogen activated T cells. Similar experiments examined the effect ofT2 on the generation of diacylglycerol. As can be seen in FIG. 9, T2inhibited IL-2 production from mitogen stimulated T cells, but had noeffect on DAG production. Additional experiments, not shown, examinedthe activity of T2 on phospholipase C activity isolated from fresh Tcells or Jurkat cells. Again, no inhibitory activity was observed. Theseexperiments suggested that the action of T2 cannot be explained by aneffect on these early signaling pathways. At these levels of T2 extractaddition, nontoxicity to other cellular functions is established asindicated by these cellular assays.

The effect of T2 on protein kinase C activation. As can be seen in FIG.10, mitogen stimulation led to translocation of PKC in Jurkat cells, andT2 did not effect PKC translocation. Finally, the effect of T2 on theactivity of protein tyrosine kinase activity was explored. As can beseen in FIG. 11, mitogenic stimulation of T cells lead tophosphorylation of a number of protein species identified with aspecific antibody to phosphotyrosine. However, T2 did not inhibit theactivity of protein tyrosine kinase since the same bands were observedregardless of the presence of T2 during mitogenic stimulation. Theseexperiments convincingly demonstrate that T2 has no effect on earlysignaling pathways involved in induction of IL-2 gene transcription.

EXAMPLE 3

In a preliminary open trial, it was found that a mixture of compounds(T2) extracted from Tripteryguim wilfordii Hook F was effective in thetreatment of rheumatoid arthritis.

To confirm the previous results obtained from these open studies, aprospective, controlled, double-blind cross-over study was designed andcarried out in the outpatient clinic of Dr. Tao in Beijing, People'sRepublic of China.

The treatment plan was designed as follows:

Seventy patients with classic or definite adult-onset rheumatoidarthritis who had active disease for more than 6 months were acceptedinto the trial and randomly assigned to 2 treatment groups. Patients inGroup A received T2 for a first course of treatment of 12 weeks, andthen were subsequently changed to placebo for a second course oftreatment of 4 weeks duration. Patients of Group B received placeboduring the first course and then were crossed-over and received T2therapy during the second course. T2 was taken in a dosage of 60 mgdaily. Placebo tablets were identical in appearance to T2 tablets. Table2 shows the treatment plan schedule.

                  TABLE 2    ______________________________________    TREATMENT PLAN (TOTAL COURSE: 16 WEEKS)                First course treat-                              Second course treat-                ment (12 weeks)                              ment (4 weeks)    ______________________________________    Group A     T2, 20 mg t.i.d.                              Placebo    Group B     Placebo       T2, 20 mg t.i.d.    ______________________________________

All patients were assessed in an arthritis clinic every 4 weeks. Theclinical assessment, overall assessment by physicians and drugdistribution were carried out by individual doctors in a blinded manner.The laboratory assessments were done by technicians of a centralhospital laboratory, who were also blinded to the details of the trial.

                  TABLE 3    ______________________________________    CLINICAL FEATURES OF PATIENTS ENTERING THE TRIAL                 First         Second                 Treatment Course                               Treatment Course                 Group A  Group B  Group A                                          Group B                 T2       Placebo  Placebo                                          T2    ______________________________________    Number of Patients                 35       35       27     31    Male/Female  3/32     4/31     1/26   4/27    Mean age, years                 46.3     48.0     46.2   47.7    Mean disease duration                 5.9      6.1      5.8    6.0    (years)    Stage of Disease    (1)          6        6        4      5    (2)          14       16       11     13    (3)          12       10       10     9    (4)          3        3        2      4    ______________________________________

The clinical features of patients entering the trial are shown in Table3. Statistical analyses demonstrated that at the beginning of the trial,Group A and Group B did not differ from each other significantly in age,sex, duration of disease or stage of disease.

                  TABLE 4    ______________________________________    RESULTS OF A CONTROLLED TRIAL OF    T2 IN RHEUMATOID ARTHRITIS             Number    No. of Patients                                  Completing Treatment             Beginning First Course                                  Second Course    Group    Treatment (12 wks)   (4 wks)    ______________________________________    A (T2 -> 35        27         24    Placebo)    B (Placebo ->             35        31         25    T2)    ______________________________________

As shown in Table 4, 27 patients of Group A completed the first courseof treatment, of which 24 completed the second course. 31 and 25 ofGroup B completed the first course and second course of treatment,respectively.

Table 5 indicates the reasons patients withdrew from the study. Threepatients of Group B but none of Group A withdrew from the trial becauseof worsening of disease during the first course of treatment, whereas 4patients from Group A but none from Group B withdrew from the trialbecause of side effects.

                  TABLE 5    ______________________________________    REASONS FOR WITHDRAWAL FROM THE STUDY                              Second               First Course Treatment                              Course Treatment               Group A  Group B   Group A                                         Group B               T2       Placebo   Placebo                                         T2               (n = 35) (n = 35)  (n = 27)                                         (n = 31)               No.    %     No.  %    No.  %   No.  %    ______________________________________    Lost to follow up               4      11    1    3    3    11  6    19    Worsening of               0      0     3    9    0    0   0    0    disease    Side effects               4      11    0    0    0    0   0    0    ______________________________________

Table 6 shows the therapeutic effects of the first course of treatment.In comparison with patients of Group B, patients of Group A showedsignificant improvement in all clinical assessments including morningstiffness, joint tenderness score, number of swollen joints, gripstrength and 15 meter walking time.

                  TABLE 6    ______________________________________    CHANGES IN CLINICAL PARAMETERS IN PATIENTS    COMPLETING THE FIRST COURSE OF TREATMENT                         Group A    Group B                         T2         Placebo                         (n = 27)   (n = 31)                                           *p    ______________________________________    Morning stiffness                 Before   2.4 ± 0.4                                     1.1 ± 0.2    (hours)      After    0.9 ± 0.2                                     2.3 ± 1.4                                           0.01    Joint tenderness                 Before  25.1 ± 1.9                                    25.5 ± 1.7    score        After    7.9 ± 1.3                                    21.9 ± 2.1                                           0.001    Number of swollen                 Before   9.2 ± 0.9                                     7.8 ± 0.7    joints       After    4.3 ± 0.6                                     7.4 ± 1.1                                           0.01    Grip strength (mean                 Before  49.0 ± 0.4                                    73.6 ± 7.7    of both sides, mm Hg)                 After   84.4 ± 7.5                                    81.2 ± 8.9                                           0.05    15 meter walking time                 Before  36.6 ± 6.6                                    37.0 ± 2.4    (second)     After   21.6 ± 1.5                                    31.9 ± 3.6                                           0.05    ______________________________________

The most noteworthy improvement was observed in joint tenderness score,which improved from a mean of 25.1 before entry to a mean of 7.9 afterthe first course of treatment with T2. By contrast, there were nosignificant changes in this score in Group B patients treated withplacebo.

As shown in Table 7, treatment with T2 also caused improvement inlaboratory correlates of disease activity. Significant improvements inESR, CRP and immunoglobulin levels were noted. The changes weresignificant at the p 0.001 level when compared between Group A and GroupB.

                  TABLE 7    ______________________________________    CHANGES IN LABORATORY PARAMETERS IN PATIENTS    COMPLETING THE FIRST COURSE OF TREATMENT                       Group A    Group B                       T2         Placebo                       (n = 27)   (n = 31)                                          *p    ______________________________________    ESR (mm/hour)               Before  69.2 ± 6.4                                  63.9 ± 5.2               After   41.0 ± 5.9                                  67.2 ± 6.6                                          <0.001    CRP (u/ml) Before  29.4 ± 5.7                                  31.6 ± 4.1               After   10.4 ± 3.9                                  43.7 ± 7.0                                          <0.001    RF (titers)               Before   87.1 ± 23.2                                   86.1 ± 35.5               After    48.0 ± 13.4                                   63.4 ± 10.9                                          NS    IgG (u/ml) Before  227.5 ± 4.6                                  231.9 ± 14.2               After   117.4 ± 9.5                                  180.4 ± 29.8                                          <0.001    IgM (u/ml) Before  302.8 ± 40.3                                  284.5 ± 32.2               After   105.2 ± 11.1                                  261.3 ± 29.3                                          <0.001    IgA (u/ml) Before  289.6 ± 29.4                                  257.6 ± 25.2               After   149.0 ± 15.5                                  280.4 ± 29.8                                          <0.001    ______________________________________     *Group A vs Group B

There was a greater tendency to decrease RF titer in T2 treated patientsbut the difference between the two groups after the first course oftreatment was not statistically significant.

During the second course of therapy, patients who had received placeboinitially improved significantly after 4 weeks of therapy with T2. (SeeTable 8). Significant improvements in joint tenderness score, number ofswollen joints and grip strength were noted. Improvement in morningstiffness and 15 meter walking time were also noted, but these changesdid not achieve statistical significance. Patients who had received T2during the first 12 weeks of therapy continued to maintain improvementeven after 4 weeks of placebo therapy during the second course.

                  TABLE 8    ______________________________________    CHANGES IN CLINICAL PARAMETERS IN PATIENTS    COMPLETING THE SECOND COURSE OF TREATMENT                      Group A        Group B                      Placebo        T2                      (n = 24)  *p   (n = 25)                                             *p    ______________________________________    Morning stiffness              Before   1.8 ± 0.2  2.5 ± 1.7    (hours)   After    0.8 ± 0.2                                NS   1.3 ± 0.9                                             NS    Joint tenderness              Before   7.9 ± 1.4  22.2 ± 2.4    score     After   11.0 ± 2.6                                NS   13.5 ± 2.0                                              <0.001    Number of Before   4.2 ± 0.8  7.0 ± 1.2    swollen   After    4.4 ± 0.9                                NS   3.5 ± 0.5                                             <0.05    joints    Grip strength              Before  87.5 ± 8.0  80.1 ± 9.2    (mean of both              After   70.2 ± 9.5                                <0.05                                     97.1 ± 13.2                                             <0.05    sides, mm Hg)    15 meter  Before  20.3 ± 1.7  31.5 ± 5.9    walking   After   17.1 ± 0.6                                NS   18.9 ± 2.3                                             NS    time (seconds)    ______________________________________     *After vs before treatment

Aside from grip strength, no significant changes were observed inclinical assessments in Group A patients after 4 weeks of placebotreatment.

As shown in Table 9, significant decreases in ESR and RF titer werenoted in Group B patients after the second course of treatment. Nosignificant worsening in laboratory parameters were noted in Group Apatients after 4 weeks of placebo therapy.

                  TABLE 9    ______________________________________    CHANGES IN LABORATORY PARAMETERS IN PATIENTS    COMPLETING THE SECOND COURSE OF TREATMENT                      Group A       Group B                      Placebo       T2                      (n = 24)  *p  (n = 25)                                            *p    ______________________________________    ESR (m/hour)              Before  42.3 ± 6.0 68.5 ± 6.9              After   31.7 ± 7.3                                NS  22.0 ± 4.9                                            <0.001    RF (titer)              Before  49.3 ± 13.5                                    67.2 ± 12.1              After   32.0 ± 12.3                                NS  32.0 ± 19.1                                            <0.05    ______________________________________     *After vs before treatment

The overall effectiveness of T2 in the present trial was classified byits capacity to induce remissions, meaningful improvement or notherapeutic effect. (See Table 10).

                  TABLE 10    ______________________________________    OVERALL EVALUATION OF THE PRESENT TRIAL                              Second               First Course Treatment                              Course Treatment    Clinical Response               Group A   Group B  Group A                                         Group B    Compared to               T2        Placebo  Placebo                                         T2    The Beginning               (n = 27)  (n = 31) (n = 24)                                         (n = 25)    of the Trial               No.    %      No.  %   No.  %   No.  %    ______________________________________    Remission  2      7.4    0    0   0    0   0    0    Improvement    Patient's  25     93     7    23  20   82  20   80    assessment    Physician's               25     93     7    23  19   79  22   88    assessment    Clinical   22     82     7    23  19   79  11   44    criteria    Laboratory 23     85     4    13  18   75  13   52    evaluation    ______________________________________

Based on the therapeutic criteria for remission in RA developed by asubcommittee of the ARA, remission was observed in two patients of GroupA at the end of the first course of treatment.

The percentage of patients who experienced meaningful improvements wassignificantly higher for Group A than for Group B patients as evaluatedby physician's assessment, and clinical and laboratory evaluations afterthe first course of treatment.

The percent of Group B patients experiencing meaningful improvementafter the second course of treatment was also remarkable, whereasimprovement was maintained in Group A patients during the 4 week secondcourse of placebo.

In order to determine whether T2 exerted an immunosuppressive effect inpatients with RA, peripheral blood mononuclear cells (PBMC) wereobtained from 18 patients of each group before and after the firstcourse of treatment. These cells were cultured for 14 days and theamounts of IgM-RF and total IgM secreted were determined using aradioimmunoassay. (See Table 11).

                  TABLE 11    ______________________________________    PRODUCTION OF IgM-RF AND TOTAL IgM BY PBMC OF    PATIENTS AFTER THE FIRST COURSE OF TREATMENT                    Group A     Group B                    T2          Placebo                    (n = 18)    (n = 18) *p    ______________________________________    RF      Before   7.2 ± 3.2                                5.4 ± 1.6            After    1.5 ± 0.5                                7.0 ± 2.2                                         <0.01    IgM     Before  220.7 ± 53.6                                260.5 ± 49.3            After   151.9 ± 55.3                                301.2 ± 100.5                                         <0.01    ______________________________________     *Group A vs Group B

In comparison with Group B, significant decreases in both IgM-RF andtotal IgM were noted in Group A after T2 treatment. These resultssuggest that T2 therapy had suppressed both IgM and IgM RF production inthese patients and thus exerted an immunosuppressive effect.

As shown in Table 12, the most common side effects of T2 were dermalreactions including skin rash, cheilosis, thinning of skin and nails andpigmentation.

                  TABLE 12    ______________________________________    INCIDENCE OF ADVERSE REACTIONS                               Second                 First Course Treatment                               Course Treatment                 Group A  Group B  Group A                                          Group B                 T2       Placebo  Placebo                                          T2                 (n = 31) (n = 31) (n = 24)                                          (n = 25)                 No.    %     No.  %   No.  %   No.  %    ______________________________________    Skin rash & cheilosis                 15     39    1    3   0    0   7    28    Diarrhea     6      27    0    0   0    0   2    8    Anorexia     2      5     0    0   1    4   0    0    Abdominal pain                 2      5     1    3   0    0   0    0    Amenorrhea   5/16   31    0    0   5/16 31  1/18 6    Postmenopausal                 1/10   10    0    0   0    0   0    0    vaginal bleeding    ______________________________________

Although the incidence of skin reactions was quite high in Group Aduring the first course of treatment, none of the patients had todiscontinue T2 treatment. Amenorrhea was another important side effectof T2. It was observed that 31% of female patients aged 49 or lesshaving received T2 for 12 weeks developed amenorrhea whereas 6% ofpatients developed it after 4 weeks of T2 treatment.

Amenorrhea disappeared in most patients when T2 was discontinued.

FIG. 12 summarizes the assessed improvements in symptoms of rheumatoidarthritis described above. T2 is an effective treatment for rheumatoidarthritis, significantly improving clinical manifestations andlaboratory correlates of inflammation. Although toxicity was frequent,it necessitated cessation of therapy in few. Clinical improvement wasobserved after only 4 weeks of therapy and persisted for at least 4weeks after the medication was discontinued. T2 therapy suppresses thein vitro production of IgM and IgM rheumatoid factor.

T2 administration has also been shown to be effective in the treatmentof systemic lupus erythematosus (Table 13). T2 appears to be effectivein relieving acute clinical manifestations including joint inflammation,skin rash and renal disease (Table 13). A steroid sparing effect of T2was also noted. In comparison with corticosteroids and commonly usedimmunosuppressive agents, such as cyclophosphamide, patients treatedwith T2 had fewer significant complications.

                  TABLE 13    ______________________________________    THERAPEUTIC EFFECT OF T2 IN LUPUS NEPHRITIS    ______________________________________    1.     Patient group           10 patients, aged 22-37, with duration of disease >1 year           were treated with T2    2.     Laboratory evaluation - before treatment           +ANA: 10           anti-DNA binding > 20%: 9           Proteinuria > 3 g/24 h: 10           Elevated serum creatinine: 3    3.     Treatment plan:           First month: T2 20 mg tid. Maintain prednisone < 40           mg/day           Followed by T2 10 mg tid. and tapered prednisone           Total course of T2: 24 weeks    4.     Results of treatment:           Serum creatinine returned to normal in 2/3           Proteinuria improved in 10/10:           undetectable: 3           <1 g/24 h: 3           >1 g/24 h: 4           Concomitant Medication           3: withdrew from prednisone           6: continued prednisone < 10 mg/day           1: changed to cyclophosphamide    ______________________________________

The following references and those cited in the text are incorporated inpertinent part by reference herein for the reasons cited in thespecification.

EXAMPLE 4 Components of T2 Extract and Toxicity Thereof

The structures of triptolide and triptodiolide are shown in FIG. 13.FIG. 14 shows the structure of triptonide. Triptolide was isolated fromalcoholic extracts of Tripterygium wilfordii Hook F by the method ofKupchan et al. (J. Am. Chem. Soc. 94, 7194-7195, 1972). This scheme fortriptolide preparation is outlined in FIG. 19.

The effect of triptolide on immunopotent cells in vitro was determinedas follows:

T cells, B cells and fibroblasts (1×10⁶ /ml) were incubated with varyingconcentrations of T2 or triptolide for 72 hr. The cells were assayed forcell viability by using a cytoflowmeter (FACSCAN) after the cells werestained with propidium iodine. Table 14 demonstrates the effect of T2 ortriptolide on cell viability.

                  TABLE 14    ______________________________________    Effect of T2 or Triptolide on Cell Viability    Inhibitors            (Percent viable cells)                    T2 (μg/ml)   Triptolide (ng/ml)    Cell type            Control 0.1    1.0  10.0 100.0                                          0.1  1.0  10.0    ______________________________________    T cells 91.7    90.0   89.3 88.2 18.8 29.5 29.8 11.5    B cells 55.6    50.9   44.3 30.5 10.6 20.9 20.9 15.6    Fibroblasts            77.5    92.7   95.1 86.6 43.0 91.7 89.3 35.8    ______________________________________

T2 at 100 μg/ml and triptolide at 10 ng/ml were toxic to fibroblastsindicating that at these levels, toxicity is nonspecific. At lowerlevels, suppression of T cell and B cell function is seen.

The capacity of triptolide to inhibit in vitro responses of humanlymphocytes was examined. As can be seen in table 15, triptolideinhibited proliferation of both T and B lymphocytes profoundly atconcentrations of 0.1-1.0 ng/ml.

                  TABLE 15    ______________________________________                 (.sup.3 H-Thymidine Incorporation, CPM)    Concentration of                 PHA-Induced T Cell                              SA-Induced B Cell    triptolide (ng/ml)                 DNA Synthesis                              DNA Synthesis    ______________________________________    0            93,400       7,900    0.1          24,200       2,000    1.0          100          100    ______________________________________

Additional experiments indicated that this triptolide fraction alsoinhibited the in vitro production of immunoglobulin from mitogenstimulated human B lymphocytes at comparably small concentrations. Theseresults support a conclusion that this triptolide fraction is extremelytoxic, however, its specificity of action is yet to be determined. Othercomponents of Tripteryguim wilfordii Hook F possibly having specificbiological activity and thought to be useful individually or incombination in the practice of the present invention include:

polpunonic acid (wilfortrine) (1) and the methyl ester thereof (2) shownin FIG. 15 and described by Keng et al. (Chem. Abst. 107:55718y, p436,1987);

triptophenolide (1) and triptophenolide methyl ether (2) shown in FIG.16 and described by Wu et al. (Chem. Abst. 107:96917f, p712, 1987);

triptonoterpenol shown in FIG. 17 and described by Deng et al. (Chem.Abst. 107:112684k, p112692, 1987); and

wilformine (shown in FIG. 18), wilforine, wilforgine, and wilforzinedescribed by He et al. (Chem. Abst. 107:130906p, 1987;

Purified components of T2 will be administered to patients withautoimmune and inflammatory diseases including rheumatoid arthritis,systemic lupus erythematosus and psoriasis. Dosage will be determinedbased on the concentration of each component in the crude T2 mixture.After phase I dosage; escalation studies are carried out to evaluatetoxicity, trials with non-toxic doses will be carried out to determineefficacy.

The following references are incorporated in pertinent part by referenceherein for the reasons cited in the specification.

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What is claimed is:
 1. A method of immunosuppression comprisingadministering Triperygium wilfordii Hook F extract in a therapeuticallyeffective amount to a patient in need of such treatment, said amountinhibiting interleukin-2 production without substantial cellulartoxicity.
 2. The method of claim 1 wherein the preparation comprisestripdiolide or triptolide.
 3. A method for suppressing autoimmunedisease consisting essentially of administering Tripteryguim wilfordiiHook F extract in a therapeutically effective amount to a patient havingan autoimmune disease, said amount inhibiting interleukin-2 productionwithout substantial cellular toxicity.
 4. The method of claim 3 whereinthe autoimmune disease is rheumatoid arthritis, systemic lupuserythematosus or psoriasis.
 5. A method of inhibiting immunoglobulinsynthesis consisting essentially of administering Tripteryguim wilfordiiHook F extract in a therapeutically effective amount to a patient inneed of such treatment, said amount inhibiting interleukin-2 productionwithout substantial cellular toxicity.
 6. A method of inhibitingcellular immune responses consisting essentially of administeringTripterygium wilfordii Hook F extract in a therapeutically effectiveamount to a patient in need of such treatment, said amount inhibitinginterleukin-2 production without substantial cellular toxicity.
 7. Amethod of selectively inhibiting the production of interleukin-2consisting essentially of administering Tripteryguim wilfordii Hook Fextract in a therapeutically effective amount to a patient in need ofsuch treatment, said amount inhibiting interleukin-2 production withoutsubstantial cellular toxicity.
 8. The method of claim 1, 3, 5, 6 or 7where the therapeutically effective amount is about 60 mg/day.
 9. Themethod of claim 1, 3, 5, 6 or 7 wherein the lack of substantial cellulartoxicity is measured by substantially unchanged interleukin-2 receptorexpression or cellular signaling activity.
 10. The method of claim 9wherein the cellular signaling activity is inositol triphosphateproduction, diacylglycerol generation, translocation of protein kinase Cor protein tyrosine kinase activity.
 11. A method for treatinginflammation or immune disease in a subject comprising:administering tothe subject a pharmacologically active amount of a Tripterigiumwilfordii Hook F root extract, the preparation having anti-inflammatoryand immunosuppressive pharmacological activity, and a steroid, whereinsaid method provides a steroid sparing effect.
 12. The method of claim11 wherein the immune disease is an autoimmune disease.
 13. The methodof claim 12 wherein the autoimmune disease is rheumatoid arthritis,systemic lupus erythematosus or psoriasis.
 14. The method of claim 11wherein the preparation comprises triptolide.
 15. The method of claim 11wherein the pharmacologically active amount is about 60 mg/day.
 16. Themethod of claim 16 wherein the steroid-sparing effect is further definedas providing pharmacological activity of the steroid at amounts lessthan capable of providing pharmacological activity in the absence of theTripteryguim wilfordii Hook F root preparation.
 17. The method of claim11 wherein the steroid is prednisone.
 18. The method of claim 11 whereinthe preparation is an ethanol extract of Tripteryguim wilfordii Hook Froot.